Ecotoxicological assessment of Green Week 2012 hospital ...

Arzneirückstände in der aquatischen Umwelt

Green Week 2012

PILLS Final Conference19th / 20th September 2012

Ecotoxicological assessment of

hospital wastewater

Dr. Nafo Folie 1

Monika Hammers-Wirtz, Henner Hollert, Issa Nafo, Sven Lyko, Axel Magdeburg, Daniel

Stalter, Jörg Oehlmann, Wilko Verweij, Evert-Jan van den Brandhof, Marja Wouterse,

Herman Evenblij Jessica Richard, Melanie Gerhards, Elke Dopp, Jochen Türk, Cornelia

Kienle, Christa S. McArdell, Colin Hunter, Dainis Sudmalis and Adrien Pilandon


• Introduction

• Objectives for bioassays in PILLS ?

• Which bioassays ?

• Which WWTP are investigated ?

Content

Dr. Nafo Folie 2

• Which WWTP are investigated ?

• Exemplary results

• Summary of results

• Conclusions


Introduction

• Hospital wastewater contains micropollutants

• Analytical detection does not allow conclusions about the potential effects on the environment

• Ecotoxicological test systems are able to integrate effects of all compounds in the sample

• Each test system gives a specific response

Dr. Nafo Folie 3

• Each test system gives a specific response

A biotest battery with different test organisms

is recommended

T47D


Objectives for bioassays in PILLS

1. Characterisation of the hospital wastewater

Comparison between hospital wastewater and municipal wastewater

2. Determination of the efficiency of the pilot plants

Comparison between raw hospital wastewater and treated hospital wastewater

Comparison between the different treatment techniques of the pilot

Dr. Nafo Folie 4

Comparison between the different treatment techniques of the pilot plants

3. Assessment of possible toxic effects induced by the advanced treatment with ozone or UV

Evaluation of effects due to by-products after ozonation or UV treatment


How are bioassays used in PILLS ?

Bioassays for specific toxicity Bioassays for non-specific toxicity

testing ...

• different mode of action

• using original and enriched samples

testing ...

• organisms of different trophic levels

• short-term and long-term exposure

• static/semistatic and flow-through design

• using original and diluted samples

Test 1...test n Test 1...test n

Dr. Nafo Folie 5

Test 1...test n Test 1...test n

Impacts on the environment


Specific toxicity

Proteins

Enzymes

Luciferase

Toxicity

Light

Dioxin

Add substrate (luciferin)

Estrogen Proteins

Enzymes

Luciferase

Toxicity

Light

Dioxin


Proteins

Enzymes

Luciferase

Toxicity

Light

Dioxin


Estrogen

• Cytotoxicity

• Mutagenicity

• Estrogenicity

• Antibiotic resistance

Which bioassays ?

Dr. Nafo Folie 6

Nucleus

Dioxinbinding

Cytosol

TranscriptionDiox inResponsiveElement(CRE)

Transport protein

Hsp

Diox in receptor

Estrogen

Estrogen

Nucleus

Dioxinbinding

Cytosol


Transport protein

Hsp

Diox in receptor

Estrogen

Nucleus

Dioxinbinding

Cytosol


Transport protein

Hsp

Diox in receptor

Estrogen

Estrogen

T47D


Which bioassays �� Specific toxicity

Bioassays Endpoints PILLS Objectives

NL* DE* CH* UK*

MTT test (with T47D cells)

Cytotoxicity (viability)

Characterisation, treatment efficiency, (oxidation by-products)

IWW with original and pre-concentrated samples

Ames test (Salmonella typhimurium,

strain YG7108)

(Reifferscheid et al. 2011)

Mutagenicity

(No. of histidine revertants)


BIK-F with original and pre-concentrated samples

Dr. Nafo Folie 7

ER Calux (with T47D cells)

Estrogenicity

(EE2 equivalent)


IWW with original and pre-concentrated samples

A-YES test (AQUA 1.0)

Estrogenicity

(Phytase activity) (EE2 equivalent)


IUTA with original and pre-concentrated samples

Waterscan (antibiotics test)

Inhibition of bacterial growth

(concentration factor EC50)


RIVM with pre-concentrated samples; Inhibition after 24 hr

*PILLS-partner countries: Germany (DE), The Netherlands (NL), Scotland (UK), Switzerland (CH)


Which bioassays ?

Non-specific toxicity �� Different trophic levels

Primary producers Secondary consumersPrimary consumers

Filter feeders

Shredders

Dr. Nafo Folie 8

DecomposersDetritus feeders


Which bioassays ?

Non-specific short-term toxicity


NL* DE* CH* UK*

Bacteria test (Vibrio fischeri)

Inhibition of luminescence (concentration factor EC50)

Characterisation, treatment efficiency

RIVM with pre-concentrated samples; Inhibition after 30 min

Ecotox Centre with pre-con-centrated samples; Inhibition after 30 min

GCU with pre-concentrated samples; Inhibition after 30 min

Algal photo-synthesis test (Selenastrum capricornutum)

Inhibition of photosynthetic efficiency (concentration factor EC50)


RIVM with pre-concentrated samples;

GCU with pre-concentrated samples;

Dr. Nafo Folie 9

capricornutum) EC50) products) d samples; inhibition after 4.5 and 24 hr

samples; inhibition after 4.5 hr

Algae growth test (Desmodesmus subspicatus)

(ISO-NWIP)

Growth rate inhibition after 72 hr

Characterisation, treatment efficiency, oxidation by-products

GAIAC with original and diluted samples

Test with Gammarus fossarum (scuds)

Mortality after 4, 24, 48, 72 hr

Characterisation Ecotox Centre with original samples

Fish embryo test (Danio rerio)

(DIN EN ISO 15088, 2009)

Mortality (coagulation of embryos, non-detachment of the tail, lack of heart-beat)

Treatment efficiency, oxidation by-products

GAIAC 48 hr, with original and diluted samples



Which bioassays ?

Non-specific long-term toxicity


NL* DE* CH* UK*

Daphnia magna population test (water flea)

(Hammers-Wirtz et Ratte 2003)

Population growth inhibition (population abundance and structure, ephippia) after 21 d

Treatment efficiency, oxidation by- products

GAIAC semi-static in laboratory with original samples

Online test with Potamopyrgus antipodarum

Mortality, shell size, number of embryos per female after 28 d

Oxidation by-products

BIK-F flow through with original

Dr. Nafo Folie 10

antipodarum (snail)

(Duff et al. 2007)

with original samples

Online test with Lumbriculus variegatus (worm)

(OECD 2007)

Reproduction, biomass, biomass per worm after 28 d

Oxidation by- products

BIK-F flow through with original samples



Country Municipal wastewater

Hospital wastewater

Germany wwtp Bad Sassendorf Marienhospital in

Gelsenkirchen

The

Netherlands

wwtp Zwolle

wwtp Hessenport

Isala Clinics, Zwolle

Wastewater treatment plants

Dr. Nafo Folie 11

Switzerland wwtp Laufäcker Hospital Baden

UK wwtp Galashiels

wwtp Shieldhall

Borders General Hospital

Western Infirmary


Results �� Municipal vs hospital wastewater

Specific effects

Effect classification

Bioassay Endpoints Raw municipal wastewater

Effluent of municipal

wastewater treatment plant

Raw hospital

wastewater

MTT test (with T47D cells) [DE]

Cytotoxicity (viability) 88.3%

(original sample)1

66.6%

(original sample)

2

A-YES test (AQUA 1.0) [DE]

Estrogenicity (EE2 equivalent)

19.7 ng/L (origninal sample)

1

43 ng/L (original sample)

2

Ames test

Dr. Nafo Folie 12

no negative effects

weak or moderate effects

strong effects

Effect classificationAmes test (Salmonella thyphimurium, strain YG7108) [DE]

Mutagenicity (No. of histidine revertants)

2

Waterscan (antibiotics test) [NL]

Sulfanomides (concentration factor EC50)

3

Waterscan (antibiotics test) [NL]

Macroliden/ ß-lactam (concentration factor EC50)

3

Origin of the samples: 1 Influent of the municipal wwtp Bad Sassendorf, Germany ( n=1, 24h-composite sample); 2

Raw

wastewater of

Marienhospital in Gelsenkirchen, Germany (n=1-4, 24h-composite sample); 3

wwtp Zwolle and wwtp

Hessenpoort, The Netherlands (n=6, grab samples)



Non-specific short-term effects – Bacteria test (30 min)

Dr. Nafo Folie 13



Non-specific short-term effects – Algal growth inhibition test (72h)

Inhib

itio

n o

f gro

wth

rate

aft

er

72

h [%

]

60

80

100

Raw hospital wastewater

Raw hospital wastewater

Raw municipal wastewater

Dr. Nafo Folie 14

G8 (12.5%) G4 (25%) G2 (50%) G1(80%)

Inhib

itio

n o

f gro

wth

rate

aft

er

72

h [%

]

-20

0

20

40

Dilution (% of wastewater)



Non-specific short-term effects

Bioassay Endpoints Raw municipal wastewater

Effluent of municipal wastewater treatment

plant

Raw hospital

wastewater

Bacteria test (Vibrio fischeri) [NL]


3

Bacteria test (Vibrio fischeri) [CH]


1.26 fold 4 0.84 fold

5

Bacteria test (Vibrio fischeri) [UK]


0.75 fold 6 0.26 fold

7

Algal photosynthesis test

Inhibition of photosynthetic

Dr. Nafo Folie 15

no negative effects


strong effects

Effect classification

photosynthesis test (Selenastrum capricornutum) [UK]

photosynthetic efficiency (concentration factor EC50)

12.07 fold 6 1.97 fold

7

Algal photosynthesis test (Selenastrum capricornutum) [NL]

Inhibition of photosynthetic efficiency (concentration factor EC50)

3

Algae growth test (Desmodesmus subspicatus) [DE]

growth rate inhibition, average for dilutions 80% and 50% wastewater

34 % 1 64-88 %

2

Test with Gammarus fossarum [CH]

Mortality after 48hr 100% 4 > 50%

5

Origin of the samples: 1 Influent of the municipal wwtp Bad Sassendorf, Germany ( n=1, 24h-composite sample); 2

Raw

wastewater of

Marienhospital in Gelsenkirchen, Germany (n=1-4, 24h-composite sample); 3

wwtp Zwolle and wwtp

Hessenpoort, The Netherlands (n=6, grab samples); 4

Raw wastewater of hospital Baden, Switzerland; 5

Influent of the

municipal wwtp Laufäcker, Switzerland ; 6 Average of influents of wwtp Galashiels (n= 9, 7d-comp. sample) and wwtp Shieldhall

(n=13, 7d-comp. sample), UK; 7 Average of raw wastewater of Borders General Hospital (n= 46, 7d-comp. sample) and Western

Infirmary (n=35, 7d-comp. sample), UK


Summary �� Objective 1

� Raw hospital wastewater was moderately cytotoxic and estrogenic, but acutely toxic to various tests organisms like bacteria, algae and invertebrates.

� In general hospital wastewater was more toxic compared to municipal wastewater.

Characterisation of hospital wastewater

Dr. Nafo Folie 16

wastewater.


Sampling points in the wwtp

Ozonation

MBR

+ O3

Sampling point

MBR

PAC reactor

Sand filter (DE)

or Moving bed

bioreactor (CH)

MBR + O3

+ SF

Pilot plants in

Gelsenkichen (DE)

and Baden (CH)

MBR

+ PAC/SFSand filter (DE)

or Moving bed

bioreactor (CH)

MBR

Dr. Nafo Folie 17

Pilot plant in

Zwolle(NL)

MBR ozonation

GAC filter

MBR + O3

+GAC

Sampling point MBR

UV/H2O2

MBR + UV

+ GAC

MBR + GAC

GAC filter

MBR

+ UV

GAC filter

MBR

+ O3


Results �� Efficiency of pilot plants

Specific effects – Ames test (No. of revertants Strain YG7108)

75

100

Mu

tag

en

icit

y [

% o

f m

ax

. re

sp

on

se

]

Dr. Nafo Folie 18N

C PC

MBR

MBR

+O3

MBR

+O3+

SF

0

25

50

Mu

tag

en

icit

y [

% o

f m

ax

. re

sp

on

se

]

� �



Specific effectsBioassays Endpoints Effluent of

MBR MBR + O3

MBR + O3 + SF

MBR + PAC/SF

MBR + O3 + GAC

MBR + GAC

MBR + UV

MBR + UV + GAC

MTT test (with T47D cells)

1

Cytotoxicity (viability)

↓ ↗ ↗

ER Calux (with T47D cells)

1


0.1 ng/L < LOD < LOD

A-YES test (AQUA 1.0)

1


0.235 ng/L 0.261 ng/L

0.176 ng/L

0.079 ng/L

Ames test

Dr. Nafo Folie 19

no negative effects


strong effects Classification

↑ ↓ in/decreasing toxicity > 20% ↗ ↘ slightly in/decreasing toxicity < 20%

Ames test (Salmonella typhimurium, strain YG7108)

1

Mutagenicity (No. of histidine revertants)

↓ ↑ ↘ ↓


2

Sulfonamides (concentration factor EC50)


2

Macrolides/ß-lactam (concentration factor EC50)

Origin of samples: 1 pilot plant in Gelsenkirchen, Germany;

2 pilot plant in Zwolle, The Netherlands



Non-specific short-term effects

Bioassays Endpoints Effluent of MBR

MBR + O3

MBR + O3 + SF

MBR + PAC/SF

MBR + O3 + GAC

MBR + GAC

MBR + UV

MBR + UV + GAC


2


↓ ↓ ↓


3


↓ ↓ ↘ ↓

Algal photosynthesis test

2

Inhibition of photosynthetic efficiency (concentration

↓ ↓

Dr. Nafo Folie 20

no negative effects




test (4.5 and 24hr)

2

(concentration factor EC50)

Algae growth test (72hr)

1

growth rate inhibition, average for dilutions 80% and 50% wastewater

↓ ↗

Test with Gammarus fossarum

3

Mortality after 48hr

variable effects

variable effects

variable effects

variable effects

Fish embryo test (48hr, DIN)

1 lethal mortality

Origin of samples: 1 pilot plant in Gelsenkirchen, Germany;

2 pilot plant in Zwolle, The Netherlands;

3 pilot plant in

Baden, Switzerland



Non-specific long-term effects

Bioassays Endpoints Effluent of MBR

MBR + O3 MBR + O3 + SF

MBR + PAC/SF

mean abundance ↗ ↘

mean of sectional growth ↗ ↗

sum of neonates

sum of dead animals

sum of aborted eggs

sum of ephippial eggs

body length on day 21

Daphnia population test (21d, semistatic)

Dr. Nafo Folie 21

no negative effects




body length on day 21

portion of adult daphnids ↗ ↘

Mortality ↗ ↗ ↓

Shell size ↑ ↓

Online test with Potamopyrgus antipodarum (28d, flow through) number of embryos/female ↑ ↘ ↑

Reproduction ↗ ↘

Biomass ↑ ↘

Online test with Lumbriculus variegatus (28d, flow though)

biomass per worm ↑ ↘

All tests performed on samples from the pilot plant in Gelsenkirchen, Germany, Evaluation is performed on average values.



Efficiency of pilot plants

� The biological treatment in a MBR considerably decreased cytotoxic and estrogenic effects in raw hospital wastewater.

� Non-specific bioassays demonstrate also a decrease in toxicity, but MBR permeate still affected some organisms like bacteria, algae and snails.

� Advanced treatment by PAC addition or GAC filtration reduced

Dr. Nafo Folie 22

� Advanced treatment by PAC addition or GAC filtration reduced ecotoxicological effects of raw wastewater, but the effluent of this treatment process still contained algal toxic compounds.


Results �� Effects after ozonation

Static test over 72h – Algal growth rate (Mean of 3 samplings)

Gro

wth

ra

te a

fte

r 7

2 h

[1

/d]

1,2

1,4

1,6

1,8

�

Dr. Nafo Folie 23

Gro

wth

ra

te a

fte

r 7

2 h

[1

/d]

0,0

0,2

0,4

0,6

0,8

1,0

MBR+O3

MBR+O3+SF

MBRControl

• MBR: 17% inhibition

• MBR+O3: 25% inhibition

• MBR+O3+SF: 24% inhibition


Online test – Reproduction of Potamopyrgus antipodarum


15

20

��

�

Nu

mb

er

of

em

bry

os [

me

an

]

Dr. Nafo Folie 24

• Increase in number of embryos after

sand filter

• OS at similar level compared to EE2

C

MBR

MBR

+O3

MBR

+O3+

SF

EE2

0

5

10

�

�

�

�

�

�

Nu

mb

er

of

em

bry

os [

me

an

]


Online test – Biomass of Lumbriculus variegatus


80

100

��

�

Dry

bio

ma

ss [

mg

]

Dr. Nafo Folie 25

• 49% reduced biomass after ozonation

• 32% after sand filter

• adverse effects in accordance with

previous studies (Stalter et al. 2011;

Magdeburg et al., 2012)

C

MBR

MBR

+O3

MBR

+O3+

SF

0

20

40

60

Dry

bio

ma

ss [

mg

]



� Ozonation reduced considerably antibiotic and estrogenic effects of hospital wastewater.

� Increasing toxicity is detected after the oxidation processes by ozonation for algae and invertebrates like daphnids, snails and worms.

Effects of ozonation by-products

Dr. Nafo Folie 26


Conclusions �� 1

� This set of bioassays is a useful tool to assess the potential effects on the environment.

� The biological treatment in a MBR decreased toxic effects considerably in raw hospital wastewater. But, MBR permeate may still affect some organisms like bacteria, algae and snails.

Dr. Nafo Folie 27

algae and snails.

� Advanced treatment by PAC addition or GAC filtration reduced the toxicity of raw wastewater, but the effluent of this treatment process may still contain algal toxic compounds.


Conclusions �� 2

� Ozonation considerably reduced antibiotic and estrogenic effects of hospital wastewater. However, increasing toxicity is detected after ozonation.

� A post-treatment of the ozonated effluent in a biofilter (e.g. sand filter with biological activity) reduced the adverse effects of the advanced treatment by oxidation significantly but did not remove it totally (e.g. toxic effects on algae).

Dr. Nafo Folie 28

but did not remove it totally (e.g. toxic effects on algae).

� A post-treatment of the UV treatment effluent by GAC filtration removed the adverse effects of the oxidation process.

� In the present study, the sand filter was not as efficient as observed at previously evaluated treatment plants to reduce oxidation product induced toxicity. Further studies on oxidation product removal after the ozonation process are desirable.


Thanks to

• Issa Nafo and Sven Lyko (Emschergenossenschaft)

• Axel Magdeburg (BiK-F), Daniel Stalter (BiK-F), Jörg Oehlmann (BiK-F), Wilko Verweij (RIVM), Evert-Jan van den Brandhof (RIVM), Marja Wouterse (RIVM), Herman Evenblij (WGS), Jessica Richard (IWW), Melanie Gerhards (IWW), Elke Dopp (IWW), Jochen Türk (IUTA), Linda Gehrmann (IUTA), Cornelia Kienle (Ecotox Centre, Eawag/EPFL), Christa S. McArdell (Eawag), Colin Hunter (GCU), Dainis Sudmalis

Dr. Nafo Folie 29

Christa S. McArdell (Eawag), Colin Hunter (GCU), Dainis Sudmalis (GCU), Adrien Pilandon (GCU) and Henner Hollert (gaiac)

• my colleagues Dennis Becker, Rachel Conrad and Melanie Grolms-Aal (gaiac)

... and you for your attention!... and you for your attention!

Ecotoxicological assessment of Green Week 2012 hospital ...

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